This invention relates to the field of vasoactive compounds and their antagonists. In particular, this invention relates to antagonists of the vasoactive peptide CGRP and other members of the CGRP superfamily.
The calcitonin gene related peptide (CGRP) is a sensory neuropeptide with potent vasodilatory and cardiotonic action as described in U.S. Pat. No. 4,530,838 to Evans et al. The peptide exists in two forms (denoted xcex1 and xcex2). xcex1-CGRP is produced by the calcitonin gene (Amara et al. Nature 298:240-244, 1982 and Rosenfeld et al. Nature 304:129-135, 1983) while xcex2-CGRP is the product of a separate gene (Amara et al. Nature 298:240-244, 1985 and Steenbergh et al. FEBS Lett. 183:403-407, 1985). The human xcex2-form and xcex1-form differ by three amino acids.
CGRP is concentrated in those areas of the body receiving sensory input from the dorsal horn with limited amounts associated with autonomic input. The peptide is present in the brain in the nuclei of sensory and motor cranial nerves and in cell bodies in the hypothalamus, preoptic area, ventromedial thalamus, hippocampus, and the like. CGRP is found in both sensory and motor nerves of the peripheral nervous system. The peptide is found in the skin, blood vessels, heart, gastrointestinal tract, tongue, esophagus, pancreas, salivary glands, lungs, kidney and other organs (Poyner, D. Pharmac. Ther. 56:23-51, 1992).
The release of CGRP from sensory nerve endings in inflammatory reactions can result in the local acceleration of microhemodynamic changes including vasodilation and permeability of the microcirculation resulting in plasma exudation and the release of humoral factors and inflammatory cells to the site of injury. CGRP has been used as a vasodilator in animal models of subarachnoid hemorrhage and in trials involving human subjects with congestive heart failure. CGRP administration produced hypotension associated with moderate tachycardia in hypertensive humans (Jian et al. Chin. Med. J 102:897-901, 1989). CGRP has also been used as a potent dilator of the coronary circulation (Ezra et al., Eur. J Pharmacol., 1987). In contrast to nitrates, which have also been used as vasodilators, CGRP results in dilation by both endothelium-dependent and endothelium-independent mechanisms. Also, in contrast to nitrates, such as sodium nitroprusside, tolerance to CGRP has not been noted Bxc3xa9ny et al. Regul. Pept. 25:25-36, 1989). CGRP has been demonstrated to improve the ability of patients to participate in exercise programs in patients with chronic stable angina (Uren et al. Cardiovasc. Res. 27:1477-1481, 1993).
CGRP has a number of problems as a therapeutic. CGRP is nonselective, inactive in oral form, generally has a short duration of action and has a number of side effects that can include uncontrolled hypotension (Feuerstein et al. Can. J. Physiol. Pharmacol. 73:1070-1074, 1995).
CGRP has been implicated in migraines, diabetes, sepsis and inflammation. Migraines are noted for the strength of the headache that ensues with its pathology. Most believe that the headache associated with migraines results from the profound cerebral vasodilation. CGRP containing nerve fibers innervate cerebral and dural vessels where CGRP is believed to prolong vasodilation. (Moskowitz Trends Pharmacol. Sci. 13:307-311, 1992). Elevated CGRP was found in the jugular vein blood of patients with migraines during a period where the patients complained of migraine symptoms, including headaches. For these reasons, CGRP antagonists have been proposed as a method for blocking cerebrovascular CGRP receptors and thus blocking the vasodilation causing migraine.
CGRP has also been postulated to be a potent indirect antagonist of insulin effects on glucose metabolism and CGRP was shown to produce insulin resistance in rat studies (Molina et al. Diabetes 39:260-265, 1990). For this reason CGRP has recently been implicated in Type II diabetes mellitus and to abnormalities associated with carbohydrate metabolism and hyperglycemia. CGRP has also been implicated in the hemodynamic derangements associated with endotoxemia and sepsis resulting from a variety of infectious diseases. Animals exposed to lipopolysaccharide (LPS) had elevated levels of CGRP and this coincided with hypotension and tachycardia in these animals (Joyce et al. Surgery 108:1097-1101, 1990 and Griffin et al. Circ. Shock 38:50-54, 1992).
CGRP binds to a number of different receptors, some of which have been characterized. Radioligand binding studies to assess CGRP affinity for CGRP receptors is well known in the literature (Poyner, D. R. Pharmac. Ther. 56:23-51, 1992). As stated in Poyner et al., a problem associated with studies to identify CGRP receptors is that lack of suitable CGRP receptor binding analogs and it is accepted that the use of CGRP antagonists is a useful way of classifying CGRP receptors. The art recognizes that there are a limited number of antagonists and that it would be desirable to have more CGRP antagonists to further classify and understand CGRP activity.
Molecules that compete for the CGRP receptor are known. These include, for example, [Tyrxc2x0]CGRP(28-37) and CGRP(8-37). Other molecules that compete for the CGRP receptor include peptides comprising the sequence of CGRP but that lack at least the first five amino acids of the CGRP amino acid sequence. [Tyrxc2x0]CGRP(28-37) was able to antagonize all forms of CGRP tested but with different potencies. Other molecules that compete for the CGRP receptor are provided elsewhere in this disclosure.
CGRP antagonists includes peptides from CGRP including amino acids 8-37 of xcex2- CGRP (Park et al. Am. J. Physiol. 1989) having the amino acid sequence: VTHRLAGLLSRSGGMVKSNFVPTNVGSKAF (SEQ ID NO: 1) and peptides from xcex1-CGRP including amino acids 8-37 and having the amino acid sequence VTHRLAGLLSRSGGMVKSNFVVPTNVGSKAF (SEQ ID NO:2) xcex2-CGRP(8-37) has been used to counteract the effects of CGRP. For example, CGRP(8-37) has been shown to reverse the hypotension and tachycardia produced by administration of LPS to rats (Huttemeir, et al. Am. J. Physiol. 265:H767-H769, 1993). In addition, CGRP(8-37) has some activity against amylin (Gardiner et al. Diabetes 40:948-951, 1991). The affinity for CGRP(8-37) varies between tissues. For example, data indicates that the affinity of CGRP(8-37) for mesenteric artery, kidney, heart and skeletal muscle is somewhat higher than the affinity of CGRP(8-37) for adipocytes and descending colon (Poyner, D. Trends in Pharm. Sci. 16:424-428, 1995).